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Protocols

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McFarlane’s Trichrome 2nd Variant for Muscle and Collagen

By Protocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step
Protocol

McFarlane's Trichrome 2nd Variant

for Muscle and Collagen

12
steps
9
materials
print

Materials

  • Weigert’s iron hematoxylin or equivalent
  • Solution A
    MaterialAmount
    Picric acid0.2g
    Acid fuchsin0.8g
    Acetic acid, glacial2mL
    Distilled water98mL
  • Solution B
    MaterialAmount
    Acetic acid, glacial2mL
    Distilled water98mL
  • Solution C
    MaterialAmount
    Picric acid1g
    Phosphotungstic acid10g
    Ethanol, 95%40mL
    Distilled water60mL
  • Solution D
    MaterialAmount
    Aniline blue2.5mL
    Acetic acid, glacial2.5g
    Distilled water97.5mL
  • Solution E
    MaterialAmount
    Picric acid0.25g
    Phosphotungstic acid2.5g
    Ethanol, 95%10mL
    Distilled water90mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with Weigert’s iron hematoxylin or equivalent.
  3. Place into solution A for 5 minute.
  4. Rinse with solution B.
  5. Place into solution C for 5 minutes.
  6. Rinse with distilled water.
  7. Place into solution D for 5-10 minutes.
  8. Rinse with solution B.
  9. Place into solution E for 5 minutes.
  10. Wash with solution B.
  11. Dehydrate with ethanol.
  12. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Erythrocytes  –  yellow
  • Cytoplasm  –  red
  • Muscle  –  red
  • Collagen  –  blue

Notes

  • McFarlane also has another multi-step method and a one step method.
  • Solutions A, C and E require dry weights of picric acid. These could be provided from saturated aqueous (1 g in 78 mL) or ethanolic (1 g in 12 mL) solutions. Alternate formulas are:

    Solution A

    MaterialAmount
    Picric acid, sat. alc.16mL
    Distilled water82mL
    Acetic acid, glacial2mL
    Acid fuchsin0.8g

    Solution C

    MaterialAmount
    Picric acid, sat. alc.12mL
    Distilled water60mL
    Ethanol, 95%28mL
    Phosphotungstic acid10g

    Solution E

    MaterialAmount
    Picric acid, sat. alc.20mL
    Distilled water70mL
    Ethanol, 95%10mL
    Phosphotungstic acid2.5g

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    McFarlane, (1944)
    Stain Technology, v. 19, pp. 29
March 30, 2023
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McFarlane’s Trichrome 3rd Variant for Muscle and Collagen

By Protocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step
Protocol

McFarlane's Trichrome 3rd Variant

for Muscle and Collagen

14
steps
9
materials
print

Materials

  • Regaud’s iron hematoxylin or equivalent
  • Solution A
    MaterialAmount
    Picric acid1g
    Orange G0.25g
    Ethanol, 95%80mL
    Distilled water20mL
  • Solution B
    MaterialAmount
    Acid fuchsin0.25g
    Ponceau 2R0.25g
    Acetic acid, glacial1mL
    Distilled water99mL
  • Solution C
    MaterialAmount
    Acetic acid, glacial2mL
    Distilled water98mL
  • Solution D
    MaterialAmount
    Picric acid1g
    Phosphotungstic acid10g
    Ethanol, 95%80mL
    Distilled water20mL
  • Solution E
    MaterialAmount
    Aniline blue2.5g
    Acetic acid, glacial2.5mL
    Distilled water97.5mL
  • Solution F
    MaterialAmount
    Picric acid0.5g
    Phosphotungstic acid5g
    Ethanol, 95%20mL
    Distilled water801mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with Régaud’s iron hematoxylin, but do not differentiate with iron alum.
  3. Place into solution A until nuclei are differentiated.
  4. Wash with water until erythrocytes alone remain yellow.
  5. Place into solution B for 5-10 minutes.
  6. Rinse with solution C.
  7. Place into solution D for 5 minutes until differentiated.
  8. Rinse with solution C.
  9. Place into solution E for 10 minutes.
  10. Rinse with solution C.
  11. Place into solution F until differentiated.
  12. Wash with solution C.
  13. Dehydrate with ethanol.
  14. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Erythrocytes  –  yellow
  • Cytoplasm  –  red
  • Muscle  –  red
  • Collagen  –  blue

Notes

  • McFarlane also has another multi-step method and a one step method.
  • Solutions A, D and F require dry weights of picric acid. These could be provided from a saturated ethanolic (1 g in 12 mL) solution. Alternate formulas are:

    Solution A

    MaterialAmount
    Picric acid, sat. alc.12mL
    Ethanol, 95%68mL
    Distilled water20mL
    Orange G0.25g

    Solution D

    MaterialAmount
    Picric acid, sat. alc.12mL
    Ethanol, 95%68mL
    Distilled water20mL
    Phosphotungstic acid10g

    Solution F

    MaterialAmount
    Picric acid, sat. alc.6mL
    Ethanol, 95%14mL
    Distilled water80mL
    Phosphotungstic acid5g

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    McFarlane, (1944)
    Stain Technology, v. 19, pp. 29
March 30, 2023
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McLachlan’s Alum Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

McLachlan's Alum Hematoxylin

6
steps
7
materials
print

This formulation has been named in honor of Dr. H.K.I. McLachlan FRCPA at the request of the originator, Mike Rentsch.

Materials

MaterialVariantFunction
Var IVar II
Hematoxylin2 g2 gDye
Aluminum sulphate17.5 g–Mordant
Ammonium alum–25 gMordant
Distilled water700 mL700 mLSolvent
Glycerol300 mL300 mLStabiliser
Sodium iodate0.2 g0.2 gOxidant
Glacial acetic acid20 mL20 mLAcidifier

Compounding Procedure

  1. Dissolve the aluminum sulphate or the Alum in about 500 mL water.
  2. Add the hematoxylin and dissolve.
  3. Add the acetic acid and mix well.
  4. Warm the glycerol to reduce viscosity, and add to the mixture.
  5. Mix well, then make up to 1 litre with water.
  6. Add the iodate and mix for 5-10 minutes.
  7. Store in a tightly capped bottle in the dark.
  8. The solution remains usable for 2-5 years.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place paraffin sections in the staining solution for 2-4 minutes and frozen sections for 40-60 seconds.
  3. Rinse with water and blue.
  4. Rinse well with water.
  5. Counterstain if desired.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • This is a progressive solution.
  • Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Rentsch, M.
    Australian Biostain P/L.
    Personal communication via the internet.
March 30, 2023
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Hitchcock Ehrich for Plasma Cells

By Intracytoplasmic Granules, Plasma Cells, Protocols, Stain Target
Protocol

Hitchcock Ehrich

for Plasma Cells

5

steps
3

materials
print

This method uses malachite green and acridine red to stain plasma cells in a similar manner to the methyl green pyronin methods.


Materials

Solution A

MaterialAmount
Malachite green1g
Distilled water100mL

Solution B

MaterialAmount
Acridine red3g
Distilled water100mL

Combine 1 part of solution A with 3 parts of solution B immediately before use.

Tissue Sample

Zenker fixation is recommended. Other fixatives may not be satisfactory.


Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into the staining solution for 30 seconds.
  3. Rinse with water.
  4. Dehydrate rapidly with absolute ethanol.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  green
  • Plasma cell cytoplasm  –  crimson
  • Other cell cytoplasm  –  pink

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.


References

  1. Biological Staining Methods, 6th ed. (1957)
    Gurr George T.,
    George T. Gurr, London, UK
March 30, 2023
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Horneyold’s Alum Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Horneyold's Alum Hematoxylin

8
steps
6
materials
print

Materials

Hematoxylin Solution

MaterialAmountFunction
Hematoxylin0.7 gDye
100% ethanol20 mLSolvent

Alum Solution

MaterialAmountFunction
Ammonium alum0.35 gMordant
Distilled water60 mLSolvent

Tincture of Iodine

MaterialAmountFunction
Iodine70 gOxidant
Potassium iodide50 g–
Distilled water50 mLSolvent
100% ethanol950 mLSolvent

Compounding Procedures

Tincture of iodine

  1. Mix the dry iodine and potassium iodide together.
  2. Add the water and mix well to dissolve.
  3. Add the ethanol and mix well.

Staining solution

  1. Dissolve the hematoxylin in the ethanol and the Alum in the water.
  2. Combine the two solutions and mix well.
  3. Leave in the light by a window for 3-4 days.
  4. Add 20 drops tincture of iodine.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 5-10 minutes.
  3. Rinse well with water.
  4. Differentiate with acetic ethanol.
  5. Rinse with water and blue.
  6. Rinse well with water.
  7. Counterstain if desired.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • Horneyold’s formula is a modification of Böhmer’s Alum hematoxylin.
  • This solution is regressive, and is recommended for osmium fixed tissue.
  • Acetic ethanol is 70% ethanol containing glacial acetic acid. The amount of acetic acid to add was not given, but 0.5%-1% should suffice.
  • Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Bolles Lee, A.. Edited by Gatenby, J.B. and Beams, H.W., (1950)
    The Microtomist’s Vade-Mecum. 11 ed.,
    Churchill, London, UK.
  2. Lange, N.A., (1966)
    Lange’s Handbook of Chemistry, Revised 10th ed.,
    McGraw-Hill.
March 30, 2023
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Lendrum’s Phloxine Tartrazine for Viral Inclusion Bodies

By Intracytoplasmic Granules, Paneth Cells, Protocols, Stain Target, Stain Type, Trichrome Staining, Yellowsolve Staining
Protocol

Lendrum's Phloxine Tartrazine

for Viral Inclusion Bodies

11
steps
6
materials
print

This method is also used for the demonstration of Paneth cell granules, and may be used as a substitute for the HPS if the differentiation in the tartrazine solution is shortened to retain pink cytoplasm and muscle.

Materials

  • Mayer’s hemalum
  • Solution A
    MaterialAmount
    Phloxine B0.5g
    Calcium chloride0.5g
    Distilled water100mL
  • Solution B
    MaterialAmount
    Tartrazineto saturation
    2-Ethoxy ethanol (cellosolve)100mL

Tissue Sample

5µ paraffin sections of formal sublimate fixed tissue is preferred. Formalin fixed tissue is suitable. Other fixatives are likely to be satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei to medium density with hemalum.
  3. Wash in tap water for 5 minutes. Blueing takes place in the phloxine solution.
  4. Place in solution A for 20 minutes.
  5. Rinse in tap water, blot almost dry. Some technologists rinse with cellosolve instead of blotting. The object is to remove all traces of water, as it interferes with the ability of tartrazine to extract phloxine and counterstain.
  6. Rinse with solution B to remove remaining water. Discard solution.
  7. Place in solution B until inclusions are red and all other tissue is yellow. The time varies considerably. Control microscopically.
  8. Rinse thoroughly but briefly with absolute ethanol.
    Do not rinse with water at this stage as it rapidly removes the tartrazine. Some technologists use cellosolve instead of ethanol.
  9. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Acidophil virus inclusion bodies  –  red
  • Paneth cell granules  –  red
  • Background  –  yellow

Notes

  • The 2-ethoxy ethanol must not be replaced by any other solvent.
  • The 2-ethoxy ethanol must be, and must remain, completely anhydrous.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Bancroft, J.D. and Stevens A. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
March 30, 2023
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Jalowy’s Impregnation for Reticulin

By Metal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type
Protocol

Jalowy's Impregnation

for Reticulin

8
steps
4
materials
print

Materials

  • Silver nitrate, 10% aqu.
  • Strong ammonium hydroxide (s.g. 0.88).
  • Ammonium hydroxide, 1% aqu.
  • Formalin, 10% aqu.

Preparation of Jalowy’s Ammoniacal Silver

  1. Place 20 mL of 10% silver nitrate in a flask and add 1 mL of 40% sodium hydroxide.
  2. Mix well, then filter. Wash the precipitate well with distilled water several times and decant.
  3. Add 20 mL distilled water to the precipitate and suspend.
  4. Add strong ammonium hydroxide by drops until the precipitate is just dissolved.
  5. Dilute to 100 mL with distilled water.

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Rinse with distilled water.
  3. Place in prewarmed ammoniacal silver solution for 5-30 minutes at 30°C.
  4. Rinse with distilled water.
  5. Rinse with 1% ammonium hydroxide.
  6. Place in 10% formalin for 2-10 minutes.
  7. Rinse well with tap water.
  8. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Background  –  brown

Notes

  • This method was recommended for collagen and reticulin fibres on formalin fixed skin.
  • Ensure that the ammonium hydroxide and sodium hydroxide are fresh and full strength. Keep the ammonium hydroxide well stoppered when not in use. Pour sufficient for use into a beaker, then immediately restopper the stock container. Do not return unused ammonium hydroxide to the stock bottle.
  • The instructions for this method do not specify to tone, fix, or counterstain. If fading or a lack of contrast make it desirable, toning with 0.1% yellow gold chloride until satisfactory, and/or fixing with 5% sodium thiosulphate, and/or counterstaining with 1% aqueous neutral red may be used.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
March 30, 2023
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Janssen’s Iron Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Janssen's Iron Hematoxylin

8
steps
6
materials
print

Materials

MaterialAmountFunction
Hematoxylin1 gDye
Ferric ammonium sulfate5 gMordant
100% ethanol5 mLSolvent
Distilled water70 mLSolvent
Glycerol15 mLSolvent
Methanol15 mLSolvent

Compounding Procedure

Gray Method

  1. Dissolve the iron alum into the water.
  2. Dissolve the hematoxylin into the ethanol.
  3. Combine, and leave for one week at room temperature.
  4. Filter, and then add the other ingredients.

Lillie Method

  1. Combines all solutions immediately once they are dissolved, and omits the week at room temperature.
  2. The solution is stable for a few months.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 15-30 minutes.
  3. Rinse with tap water.
  4. Dip briefly in 1% hydrochloric acid in 70% ethanol.
  5. Wash well in running tap water to blue.
  6. Rinse with distilled water.
  7. Counterstain if desired.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Background  –  as counterstain or unstained

Notes

  • This solution may be used as an acid resistant nuclear stain.
  • It is recommended as a substitute for Weigert’s iron hematoxylin.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    Janssen, (1897),
    Cellule, v.14, p.207.
    Lillie & Earle, (1939),
    Stain Technology, v.14, p.53
March 30, 2023
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Kefalas’s Iron Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Kefalas's Iron Hematoxylin

16
steps
4
materials
print

Materials

Solution A

MaterialAmountFunction
Hematoxylin1 gDye
Ferric chloride1 gMordant
Acetone100 mLSolvent
Hydrochloric acid0.05 mLAcidifier

Compounding Procedure

  1. Dissolve the ferric chloride and hematoxylin in the acetone.
  2. Add the hydrochloric acid.

Protocol

Standard Method

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 30 minutes.
  3. Wash well in running tap water to blue.
  4. Rinse with distilled water.
  5. Counterstain if desired.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Alternative Method

Since this solution is almost anhydrous it can be used in a staining procedure which completely avoids water or ethanol.

  1. Remove wax with xylene.
  2. Remove xylene with a few changes of acetone.
  3. Place into staining solution for 30 minutes.
  4. Wash with acetone.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Background  –  as counterstain or unstained

Notes

  • 0.05 mL hydrochloric acid would be about 1-2 drops.
  • The simplicity of the formula indicates it is likely not stable for long.
  • The presence of hydrochloric acid may indicate progressive staining.
  • It is likely suitable as an acid resistant nuclear stain.
  • Although the staining time should be established by trial and error, 30 minutes would likely suffice as a starting point.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    Kefalas, (1926),
    Journal of the Royal Microscopical Society
    v. 46, p. 277.
March 30, 2023
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Kiernan’s Eriochrome Cyanin for Nuclei

By Dye Type, Hematoxylin Alternatives, Protocols, Stain Type
Protocol

Kiernan's Eriochrome Cyanin

for Nuclei

8
steps
6
materials
print

This is a substitute for alum hematoxylin in the H & E stain.

Materials

Solution A

MaterialAmount
Eriochrome cyanine R1.0g
Ferric chloride, 5.6%20mL
Sulphuric acid, conc.2.5mL
Distilled waterup to 500 mL

Solution B

MaterialAmount
Hydrochloric acid, conc.5mL
Ethanol, abs.500mL
Distilled water500mL

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place sections into solution A for 5 minutes.
  3. Wash with running tap water for about 30 seconds.
  4. Differentiate with solution B until nuclei are sharply stained, about 10-30 seconds.
  5. Wash with running tap water for about 5 minutes.
  6. Counterstain with eosin or another contrasting dye, as wished.
  7. Dehydrate with ethanol.
  8. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstained

Notes

  • Solution A is stable for years.
  • Solution A is also used for Kiernan’s myelin staining variant.
  • Kiernan also describes a two colour staining variant.
  • Eriochrome cyanine R is also known as mordant blue 3 and solochrome cyanine R.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Kiernan. J.A., (1999)
    Histological and histochemical methods: Theory and practice, Ed. 3
    Butterworth Heinemann, Oxford, UK.
March 30, 2023
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